Homologous recombination in mammalian cells
First Claim
1. A method for generating a DNA segment inside a cell comprising the step of introducing at least a first and a second DNA fragment into a recombination competent mammalian cell, each of said DNA fragments having a 5'"'"' and a 3'"'"' sequence portion, said first DNA fragment 3'"'"' sequence portion having at least 80% sequence identity to said second DNA fragment 5'"'"' sequence portion, wherein said first and second DNA fragment homologously recombine in said cell to form are combined DNA segment greater than 50 kb in length, said recombined DNA segment being integrated into the genome of said cell.
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Accused Products
Abstract
The invention relates to methods for intracellularly producing DNA segments by homologous recombination of smaller overlapping DNA fragments and transgenic mammalian cells and transgenic non-human mammals produced by such methods.
330 Citations
23 Claims
- 1. A method for generating a DNA segment inside a cell comprising the step of introducing at least a first and a second DNA fragment into a recombination competent mammalian cell, each of said DNA fragments having a 5'"'"' and a 3'"'"' sequence portion, said first DNA fragment 3'"'"' sequence portion having at least 80% sequence identity to said second DNA fragment 5'"'"' sequence portion, wherein said first and second DNA fragment homologously recombine in said cell to form are combined DNA segment greater than 50 kb in length, said recombined DNA segment being integrated into the genome of said cell.
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8. A method for generating a DNA segment inside a cell comprising the step of introducing at least a first and a second DNA fragment into a recombination competent mammalian cell, each of said DNA fragments having a 5'"'"' and a 3'"'"' sequence portion, said first DNA fragment 3'"'"' sequence portion having at least 80% sequence identity to said second DNA fragment 5'"'"' sequence portion, wherein said first and second DNA fragment homologously recombine in said cell to form a recombined DNA segment of at least 50 kb integrated into the genome of said cell, and said DNA segment does not encode a positive selection marker which can be used to detect homologous recombination between said first and said second fragments.
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9. A method for generating a DNA segment inside an embryonic cell comprising the step of introducing at least a first and a second DNA fragment into a mammalian embryonic cell, each of said DNA fragments having a 5'"'"' and a 3'"'"' sequence portion, said first DNA fragment 3'"'"' sequence portion having at least 80% sequence identity to said second DNA fragment 5'"'"' sequence portion, wherein said first and second DNA fragment homologously recombine in said embryonic cell to form a recombined DNA segment of at least 50 kb, said recombined DNA segment being integrated into the genome of said embryonic cell.
- 11. A transgenic mammalian cell comprising a transgene longer than about 50 kb integrated into the genome of the cell wherein the transgene expresses a protein at a detectable level, and the mammalian cell is a non-human zygote or a non-human mammalian embryonic stem cell capable of populating the germline of a mammal.
- 12. A transgenic non-human mammal containing a transgene integrated into the genome of the mammal, comprising a recombined DNA segment of at least 50 kb formed by homologous recombination of a first and a second DNA fragment, each of said DNA fragments having a 5'"'"' and a 3'"'"' sequence portion, said first DNA fragment 3'"'"' sequence portion having at least 80% sequence identity to said second DNA fragment 5'"'"' sequence portion, wherein the transgene expresses a protein at a detectable level.
- 17. A transgenic nonhuman mammal comprising a transgene longer than 50 kb integrated into the genome of the mammal, wherein the transgene expresses a protein at a detectable level.
Specification